The role of costimulation in T cell repertoire selection has been studied. Mice with genetically altered expression of costimulatory molecules CD80, CD86, and CD40, or of the costimulatory receptors CD28 and CD154 are being analyzed for thymic development and expressed T cell repertoire. We have demonstrated that expression of CD40L (CD154) is critical to negative selection of self-reactive T cells. Two pathways of negative selection have been identified. One pathway is CD40L-dependent and acts at a relatively early stage in intra-thymic development and is mediated by a non-cell-autonomous mechanism. The second is CD40L-independent and occurs later in intra-thymic or post-thymic development. Interestingly, we have observed that when negative selection is abrogated by inactivation of the CD40L pathway, functional self tolerance is maintained by a non-deletional mechanism. This non-deletional tolerance is mediated by a CD28-dependent pathway. Thus, redundant pathways exist to protect against self reactivity in the thymus, and disruption of both C40L (deletional) and CD28 (non-deletional) mechanisms of self tolerance results in a population of highly self-reactive thymic T cells. We have also demonstrated a requirement for CD28 costimulation in the induction of both deletional and anergic tolerance to male-specific antigens in pregnant male antigen-specific TCR-transgenic mice. Together, these findings have elucidated the roles of costimulatory pathways in T cell repertoire selection and maintenance of self tolerance. The expression of both CD28 and B7 is highly regulated during thymic development, such that the most immature thymocytes are protected from exposure to costimulatory signaling. We hypothesized that costimulation of immature thymocytes might disrupt normal differentiation, and addressed this hypothesis by genetically engineering ectopic expression of costimulatory receptor/ligand molecules. The transgenic over-expression of CD28 and CD86 had dramatic effects on thymic maturation, leading to differentiation of CD4+CD8+ thymocytes in the absence of TCR expression, suggesting a previously undescribed ability of costimulatory signaling to drive T cell development, and the critical need to regulate costimulation during thymic development in order to avoid activation of an abortive differentiation pathway.